13 5 Vol13 No5 2009 10 Life Science Research Oct 2009 2009 *,, 200040 :,, GFP, GFP, : ; ; ; ; : Q331 :A :1007-7847(2009)05-0394-05 A Novel Method for the Determination of Recombinant Lentiviral Titer and Infectivity by qrt- MA Hai-yan FANG Yu-dan ZHANG Jing-zhi * School of Medicine Shanghai Jiaotong University Shanghai Children s Hospital Shanghai Institute of Medical Genetics Shanghai 200040 China Abstract Lentiviral vector is being widely used in the study of gene therapy in animal models and in generating transgenic animals However determination of lentiviral particles and their infectivity is essential before their being used Such a requirement can be accurately achieved by qrt- Refered by infectious units got from GFP reporter assay it showed a positive correlation between the two approaches A reliable accurate and rapid method is therefore established for the determination of the recombinant lentiviral titer and the infectivity Key words lentiviral vector qrt- viral titer integration copy number infectivity Life Science Research 2009 13 5 394~398 [1~3] [4] 8 kb [5] HIV-1 MuLV 5 [6] : 2009-03-18 : 2009-05-16 : 2007AA021206 30870943 08ZR1412100 : 1983- * 1959- Tel 021-62472308 E-mail jzhang38@hotmailcom
5 : 395 2 μl DNase 5 μl 10 DNase I Buffer 2 μl DNase I GFP 5 U / ul Takara Bio Inc Shiga Japan 2 μl p24 - ELISA RNase inhabitor Takara Bio Inc Shiga Japan DEPC 50 μl 37 45 min DNase 350 μl STE 20 μl 10% SDS 5 μl K 20 g / L AMRESCO Solon OH 56 15 min 20 μl DEPC 14 LTR RNA / LTR DNase I DNA 1 μg RNA 20 pmol RT- GFP 5 -GAGAGCTCCCAGGCTCAGATC-3 2 μl 5 RT Buffer 1 μl MLV Takara Bio Inc Shiga Japan 10 μl 37 1 h 15 LTR 1 900 1 nmol / L 250 nmol / L 25 μl 10 Ex-Buffer 11 3 FUGW 5 μl 25 μl egfp 89 Corbett Life Science RG-3000 Sidney VSVG Australia 95 5 min 95 30 s Marine Medical Center Research Institute Promega ProFection Kit FUGW 15 μg 89 10 μg VSVG 75 μg 6 h 10% 001 0001 μl FBS DMEM 37 5% CO 2 60 h 13 RNA 15 μmol Mg 2 + 25 μmol dntp 1 U ExTaqE 59 30 s 40 59 520 nm 293T ATCC American Type Culture Collection 1 ProFection Promega Table 1 The sequences of primer and probe of Realtime FBS Hanks Salmon Sperm DNA Primer Sequences Gibco BRL STE 10 mmol / L Tris ph LTR-F 5 -ACAGCCGCCTAGCATTTCAT-3 76 1 mmol / L EDTA ph 80 01 mol / L NaCl LTR-P 5 -GAGAGCTCCCAGGCTCAGATC-3 12 FUGW LTR-Probe 5 -ACATGGCCCGAGAGCTGCATCC-3 293T 70% 16 FUGW 293T 10 01 632 10 7 632 10 6 50 000 g 632 10 5 8 mg / L Polybrene 15 h Sigma-Aldrich Inc St Louis MO Hanks -80 293T 15 10 6 / 37 2 h 2 d
396 2009 2 17 DNA 2 d 21 15 ml EP tube 200 g 5 min LTR 200 μl STE 20 μl 10% SDS 10 μl K 37 4 h LTR 2 15 000 g 12 min LTR 15 000 g 126 10 12 / ml 6 min 1/10 3 mol/ml 1 2 NaAc -20 1 h 632 10 11 / ml 15 000 g 4 20 min 100 μl TE 18 293T DNA LTR 15 GFP Norm fluore 10^-1 10^-2 Threshold Threshold cycle 40 30 20 10 R=0999 80 R^2=0999 60 Efficiency=096 M=0293 B=13087 10^-3 0 5 10 15 20 25 30 35 Cycle number 1 Fig1 Fluorescence intensity curve of Real-time 0 10^4 10^5 10^6 10^7 10^8 10^9 Concentration Copy number 2 Fig2 Standard curve of Real-time 22, =LTR / 4 159 ± 064 10 10 IU / ml 10 23 GFP 01 001 0001 μl 15 10 6 01 001 0001 μl 293T 2 d 2 d DNA LTR GFP 01 001 01 001 0001 μl 0001 μl GFP 532 10 6 52% 6% 05% 3 928 10 5 448 10 4 = GFP R = 099 R 2 = 099 Efficiency = 101 810 ± 079 10 9 IU/mL 24 GFP DNA, LTR GFP
5 : 397 2 GFP GFP A B C 3 293T GFP (A) 01 μl ; (B) 001 μl ; (C) 0001 μl Fig3 GFP expression in infected 293T cells after 10 fold dilution A 293T cells infected by 01 μl virus B 293T cells infected by 001 μl virus C 293T cells infected by 0001 μl virus 2 GFP Table 2 Comparison of lentivirus titer determined by GFP reporter assay and qrt- approach Dose of viral particles 63 10 7 63 10 6 63 10 5 Percentage of GFP + cells 520% 60% 05% GFP reporter assay Infectious units 78 10 5 90 10 4 75 10 3 qrt- assay Infectious units 13 10 6 23 10 5 11 10 4 293T 15 10 6 Notes 293T cells in each well are: 15 10 6 [8~10] 25 = / 100% 265 ± 107% 3 [11,12] [13] [14] HIV-1 HIV-1 1 p24 p24 ELISA ELISA 6 000 [7] HIV-1 2 3
398 2009 LTR : (References): 1 [1] WU X LI Y CRISE B et al Transcription start regions in the human genome are favored targets for MLV integration[j] Science 2003 300 1749-1751 [2] DEPALMA M MONTINI E SANTONIDESIO F R et al LTR 2 GFP hematopoietic cells[j] Blood 2005 105 6 GFP hotspots[j] Cell 2002 110 521-529 GFP 3 Neurosci Res 2003 73 6 876 GFP [J] Jing-zhi GUO Xin-bin XIE Shu-yang et al Promoter trapping reveals significant differences in integration site selection between MLV and HIV vectors in primary 2307-2315 [3] SCHRODER A R SHINN P CHEN H et al HIV-1 integration in the human genome favors active genes and local [4] JAKOBSSON J ERICSON C JANSSON M et al Targeted transgene expression in rat brain using lentiviral vectors[j] [5] Science 2006 16 8 827-832 cells[j] RNA 2003 9 4 493-501 [7] LIU Yin transgenic animal development[j] Int J Genet 5 374-377 9007-9011 93 3 357-362 lentiviral vectors[j] Science 2002 295 868-872 293T 4% embryonic stem cells and preimpalntation embryos[j] Natl Acad Sci U S A 2002 99 2140-2145 EMBO Reports 2003 4 1054-1060 ZHANG Production of transgenic mice carrying green fluorescence protein gene by a lentiviral vector-mediated approach[j] Progress in Nature [6] STEWART S A OYLOCHOOM D M PALLISER D et al Lentivims-delivered stable gene silencing by RNAi in primary [J] Application of lentiviral vectors in 2007 30 [8] MAY C RIVELLA S CALLEGARI J et al Therapeutic haemoglobin synthesis in beta-thalassaemic mice expressing lentivirus-encoded human beta-globin[j] Nature 2000 406 6791 82-86 [9] HAN X D LIN C CHANG J et al Fetal gene therapy of α-thalassemia in a mouse model[j] PNAS 2007 104 21 [10] LI W XIE S Y GUO X B et al A novel transgenic mouse model produced from lentiviral germline integration for the study of β-thalassemia gene therapy[j] Haematologica 2008 [11] LOIS C HONG E J PEASE S et al Germline transmission and tissuespecific expression of transgenes delivered by [12] PFEIFER A IKAWA M DAYN Y et al Transgenesis by lentiviral vectors lack of gene silencing in mammnlian Proc [13] HOFMANN A KESSLER B EWERLING S et al Efficient transgenesis in farm animals by lentiviral emtovora vectors[j] [14] HOFMANN A ZAKHARTCHENKO V WEPPERT M et al Generation of transgenic cattle by lentiviral gene transfer into oocytes[j] Biol Reprod 2004 71 405-409